First record of the exotic Indothais lacera (Gastropoda, Muricidae) in Brazil

Abstract Bioinvasions are one of the main causes of the decline of native biodiversity. Indothais lacera (Born, 1778) is a carnivorous gastropod, native to the Indo-Pacific. We present the first records of I. lacera on the Brazilian coast and the first record of this invasive species in the Western Atlantic. The specimens were found in two locations in the Baía de Todos os Santos, Bahia state, northeast of Brazil. Live specimens were collected in the Itapagipe Peninsula, in Ribeira, Salvador, and dry samples (empty shells) were collected in the extreme north of Itaparica Island. Molecular analyses indicate that there was more than one event of introduction of I. lacera in the bay and alert to the potential impacts that this species can have on the benthic community in the region. We suspect that this exotic species is affecting a native population of Thaisella, which was previously locally abundant but has since become rare.


INTRODUCTION
Bioinvasions have become a growing concern in recent years due to the potential threat that the introduction of exotic species poses to native biodiversity.In environments profoundly altered by humans, bioinvasions can lead to a substantial change in the structure and composition of native communities, causing significant changes in natural ecosystems (Ruiz et al., 1997;Byers, 2002).
In estuaries, bays and other coastal environments, the inadvertent introduction of exotic species, mediated by anthropogenic actions, is mainly related to activities of socio-economic interest, such as maritime transport and aquaculture.As a result, transfer via ballast water in ships has acted as the main vehicle in the dispersion of bioinvaders across coastal environments (Wonham et al., 2001;Silva & Barros, 2011).For mollusks, in particular, Brazil has not been an exception, and several recent discoveries have been made in coastal marine environments, including gastropods, bivalves and a chiton (Simone & Gonçalves, 2006;Breves-Ramos et al., 2010;Rocha et al., 2013;Oliveira et al., 2017;Spotorno-Oliveira et al., 2018, 2020;Amaral et al., 2019;Gernet et al., 2019;Belz et al., 2020;Jardim & Pedro, 2021).
In a study dealing with Western Atlantic estuarine muricids, focusing on the genus Thaisella Clench, 1947 (Rapaninae), an anomalous population has been found in the region of Baía de Todos os Santos, Bahia state, Brazil.The shells have a tall spire with highly shouldered, even carinated whorls, with peripheric tubercles in the form of spirally elongated blades.These characters are unusual in Thaisella, but are found in the related Indo-Pacific genus Indothais Claremont, Vermeij, Williams & Reid, 2013.Both Thaisella and Indothais share the estuarine habitat and a well-developed thread flanking the umbilical area of the shell.In the genus Indothais, the type species I. lacera (Born, 1778) has already been recorded outside its native range, namely in the Eastern Mediterranean (Gofas & Zenetos, 2003).This shallow-water muricid is native to the Indo-Pacific, occurring from the Arabian Peninsula to Southeast Asia (Rosenberg, 2006;Kumar et al., 2015;Kantharajan et al., 2017;Niamaimandi et al., 2017;Zhong et al., 2017;Abu-Zied & Bantan, 2018;Jeeva et al., 2018;Mahapatro et al., 2018;Al-Asif et al., 2020;Sultana et al., 2021).It has a biconical shell, with strong spiral cords, whorls often bearing strong triangular tubercles, aperture ovate and a broad and apparent pseudoumbilicus (Kumar et al., 2017).
The conchological and molecular analysis of specimens from the anomalous population from Baía de Todos os Santos demonstrates that they actually belong to I. lacera.Thus, a new exotic species is reported here from the Brazilian coast, being likewise the first report from the Western Atlantic.We also discuss its possible mode of transportation and the potential implications of its introduction to Brazilian coastal ecosystems.

DNA extraction, amplification and sequencing
Selected ethanol-preserved specimens (three from lot MZSP136193 and two from lot MZSP149013) had a small section of their foot clipped for DNA extraction.The extraction was conducted following the standard protocol of the QIAGEN DNEasy® Blood & Tissue Kit, repeating the final step to increase the yield.
Two mitochondrial markers were targeted for this study: the circa 650 bp long barcoding fragment of the mitochondrial COI (cytochrome c oxidase subunit I) gene, using the primers LCO and HCO of Folmer et al. (1994); and the circa 450 bp long 16S rRNA (large subunit rRNA) gene, using the primers 16SarL and 16SbrH of Simon et al. (1994).The protocol for PCR amplification for both markers was: initial denaturation at 96℃ (3 min); 35 cycles of denaturation at 95℃ (30 s), annealing at 48℃ (1 min), and extension at 72℃ (2 min); final extension at 72℃ (5 min).The success of the PCR was assessed via agarose gel electrophoresis and the PCR products were cleaned using ExoSAP-IT™ (Affymetrix Inc.) according to the manufacturer's protocol.The samples were sent to Massey Genome Service (Massey University, Palmerston North, New Zealand) for Sanger sequencing.The resulting sequences were quality-proofed and assembled in Geneious Prime (v.2020.2.2, Biomatters Ltd.), and the consensus sequences were uploaded to GenBank (see Table 1 for registration numbers).

Phylogenetic analysis
To confirm the identity of the Brazilian specimens, a phylogenetic analysis was conducted including GenBank sequences of the species of interest (Indothais lacera), as well as seven further Indothais spp.and four other species belonging to related Rapaninae genera (Table 1).The Muricinae species Murex pecten [Lightfoot], 1786 was used as an outgroup.
The alignment of the sequences of each marker was done in Geneious Prime using the MUSCLE plugin (Edgar, 2004) with default settings (i.e., optimized for accuracy).The alignments were visually proofed for inconsistencies and then run through Gblocks (Castresana, 2000;Talavera & Castresana, 2007) with the least restrictive settings, to eliminate poorly-aligned or data-deficient positions that could interfere with the analysis.The resulting COI and 16S alignments were then concatenated for the phylogenetic analysis.The Bayesian inference phylogenetic analysis was done using MrBayes (v.3.2.7, Ronquist et al., 2012) via the CIPRES Science Gateway (v.3.3, Miller et al., 2015).Two concurrent analyses were run, each with 4 Markov chains of 30 million generations (the first 20% of generations were discarded as 'burn-in'), the default priors, nst = 6, rates = invgamma, temperature parameter = 0.1, sampling every 1,000 generations, and with substitution model parameters unlinked across the two markers.MCMC convergence was assessed by the standard deviation of split frequencies (< 0.01) and the potential scale reduction factor (PSRF ~ 1.0), as well as by observing the trace plots in Geneious (Ronquist et al., 2009).

RESULTS
The specimens of Indothais from the Brazilian coast exhibit shell features consistent with those of I. lacera, which are: a biconical shell with strong spiral cords, the presence of a ridge on the sutural ramp, whorls provided of strong triangular tubercles, aperture ovate and a broad and apparent pseudoumbilicus (Tan & Sigurdsson, 1996;Kumar et al., 2017).
In total, 31 terminal taxa were used in the molecular phylogenetic analysis, including the outgroup (Table 1).After the exclusion of poorly-aligned or data-deficient positions with Gblocks, the resulting COI sequences were 654 bp long, and 16S were 445 bp long, resulting in concatenated sequences 1099 bp long.The Bayesian analysis obtained a well-resolved tree, with mostly well-supported nodes throughout (posterior probabilities, PP ≥ 0.95; Fig. 3).
The sequences of I. lacera are all gathered in a single clade with full support (PP = 1), which includes all specimens collected on the Brazilian coast (Fig. 3).Three of the Brazilian specimens form a strongly-supported (PP = 1) monophyletic taxon that is sister to all other I. lacera.The other two specimens form another strongly-supported (PP = 0.99) monophyletic branch that is sister to a clade formed by two specimens of I. lacera from Bangladesh (PP = 1).This confirms the identity of the Brazilian specimens as I. lacera and also indicates that the population of this species on the Brazilian coast could have more than one origin, i.e., it was introduced more than once, from different localities.
Another result of note, albeit unrelated to the present question, is that the specimens of I. javanica from China are potentially misidentified specimens of I. rufotincta (Fig. 3).The description and diagnostic features of I. javanica have been flagged as potentially problematic in other recent studies that made use of both morphological features and molecular data (Marshall & Taha, 2021).

DISCUSSION
Baía de Todos os Santos is the second largest coastal bay in Brazil, covering a maximum area of 1,233 km² (Cirano & Lessa, 2007).That bay is located in the northeast region of the country, close to the city of Salvador, the capital of the state of Bahia, and has ten large port terminals (Hatje & Andrade, 2009), which means intense maritime activities in the region.Judging by the high port activity in the bay, it is expected that the dispersion of I. lacera to Brazil has occurred through inter-oceanic travel.This can take place either through ballast water (the species has a long planktonic larval stage) or biofouling (i.e., as an epibiont on ship hulls).
Considering that the Brazilian specimens of I. lacera form two distinct clades in the resulting phylogenetic tree (Fig. 3), it can be surmised that there was more than one introduction event of this species in Brazil, from at least two different localities.One of these localities is the region around Bangladesh, given the small genetic distance between them (Fig. 3).The remaining specimens from Brazil display a greater genetic distance from other sequenced specimens of I. lacera from China and Malaysia (Fig. 3).Given the absence of sequenced specimens from additional localities, it is presently not possible to narrow down the locality of origin for the second introduction of I. lacera in Brazil.Nevertheless, given that each branch of Brazilian I. lacera on the tree contains specimens of the two localities sampled, it can be assumed that the populations from each introduction event have spread throughout the bay.
Rapaninae species are known to be generalist predators of other molluscs, exerting great influence on the structure of faunal communities (Vermeij & Carlson, 2000).Consequently, this invasion may pose a potential risk to native mollusc assemblages in the region.Although rapanines are widely known to inhabit rocky shores and reef ecosystems, the broad hyaline tolerance of I. lacera (Claremont et al., 2013) may also signify a risk to estuarine ecosystems.This fact may be related to the apparent disappearance of the native species Thaisella mariae (Morretes, 1954) on Itaparica Island (NCP, unpublished data), although it is impossible to ascertain this at present.Thaisella mariae is a muricid endemic to Brazil that occurs in estuarine areas, with a northern limit in Bahia state (Itaparica) and a southern limit in Paraná state (Rios, 2009).This species has a fusiform shell, relatively smaller in size compared to the invasive species (about 30 mm), characterized by sharp nodules that form fissured needles (although quite variable), a wide umbilical area surrounded by a thick keel, and by the presence of 3 to 4 bands of colour ranging from bluish-purple to brown inside the outer lip.However, in recent collections (2018) carried out in the region, no evidence of its occurrence was found.As specimens of T. mariae were relatively common in those areas, it is possible to speculate that the native species may have been displaced and locally extinct by the Indo-Pacific exotic species (T.mariae apparently occupies the same niche as I. lacera).
Although the real impact of this invader in Baía de Todos os Santos is only speculative for the moment, its introduction (from at least two source populations) is a reminder that there are still few efforts dedicated to the prevention of marine bioinvaders in Brazilian ports (e.g., Gernet et al., 2019;Spotorno-Oliveira et al., 2020).Considering the ecological importance of molluscs within the marine benthic fauna, a significant change in the species composition of mollusc assemblages could have cascading effects on the entire benthic community, especially considering a fauna that already suffers from the impacts generated by port activity (Hatje & Andrade, 2009).Considering that bays and other coastal regions with intense port activity are frequent sites of marine invasions (Ruiz et al., 1999), stricter and more optimized methods of controlling and preventing bioinvasions in those areas are urgently needed (Darrigran et al., 2020).

Figure 1 .
Figure 1.Baía de Todos os Santos and sites where Indothais lacera was found: 1 = Itaparica Island; 2 = Itapagipe Peninsula.The other symbols indicate the major ports in the region: square = Port of Aratu; circle = Port of Salvador.

Figure 3 .
Figure 3. Bayesian inference phylogenetic tree based on CO1 and 16S, with the specimens collected in Brazil highlighted.Posterior probabilities are shown on nodes; scale bar is substitutions per site.

Table 1 .
List of species used in the phylogenetic analysis (including our specimens), with GenBank registration numbers of the COI and 16S markers, and provenance data of the specimens sequenced.